Helix-coil Transition Temperature Research Articles

New insights into xanthan synergistic interactions with konjac glucomannan: A novel interaction mechanism proposal

The interactions of xanthans containing precise acetate and pyruvate concentration with Konjac glucomannan (KGM) were studied at different sodium chloride and polymer concentrations. A new unified model of the interaction is proposed, taking into account previous models in the literature. This study suggests that the interactions occur by two distinct mechanisms dependent on xanthan conformation. These interactions are not mutually exclusive and may co-exist and hence produce complicated traces. Consequently two types of gel which melt at different temperature ranges can be formed. Depending on the xanthan helix coil transition temperature, one or both of the synergistic states may exist in the hydrocolloid blend. The proposed model has been tested rheologically and using differential scanning calorimetry by varying salt concentration and using samples containing different functional group concentrations.

Possible mechanism of non-monotone behavior of helix-coil transition temperature in two-component solvent

Within the frameworks of generalized model of polypeptide chain (GMPC) the concentrational behavior of helix-coil transition temperature of biopolymer in two-component solvent was observed on the simplest models. It is shown that if the solvent and the ligand interact with the biopolymer at different binding sites, and even if both components influence the helical state in the same way, the model allows one to obtain the non-monotone character of the melting temperature behavior of biopolymer depending on the ligand concentration. It is shown that changes in the regime of helical state stability is not always related with the competition of stabilizing and destabilizing interactions of blend components but can be the result of the cumulative effect of both components.

Specific features of Zn2+, Co2+ and Ni2+ ion binding to DNA in alkaline solutions

Dependence of DNA metallization degree during B-DNA transition into the metallized (m) form on DNA concentration has been studied by visible and differential UV-spectroscopy in the presence of Zn2+, Co2+ and Ni2+ ions in tetraborate buffer (pH 8.5) with and without ethidium bromide. Constants of Mt2+ binding to double stranded DNA were calculated. The obtained binding constants corresponded to the formation of inter-strand metal bridges stabilizing m-form. Thermodynamic origin of higher efficiency of Zn2+ ions in DNA metallization compared to Co2+ and Ni2+ was revealed. Increase of the DNA helix-coil transition temperature by up to 10°С upon formation of m-form in the presence of Zn2+ ions was observed and rationalized. Furthermore, a strong cooperative decrease (up to 30°С) of the temperature of В→m transition induced by heating in the presence of Zn2+ was found and its nature was explained.

Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding

The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c 6 in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1–79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His 6 tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I Ref. [5] and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50–65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and 15N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.

The effect of hydrostatic pressure on the thermal stability of DNA hairpins

DNA hairpins consist of two distinct structural domains: a double stranded stem and a single-stranded loop that connect the two strands of the stem. Previous studies of short DNA hairpins have revealed that loop and stem sequences can significantly affect the thermodynamic stability of short DNA hairpins. In this work we present the effect of hydrostatic pressure on the helix-coil transition temperature (T M) for 11 16-base, hairpin-forming DNA oligonucleotides. All of the samples form a hairpin with a 6-base pair stem and a four-base loop. In addition, the four base pairs at the end of the stem distal from the loop are the same for every molecule. We have varied loop sequence and identity of the two duplex base pairs adjacent to the loop. Using the change in UV absorption to monitor the conformational state of the oligonucleotide the hairpin-coil transition temperature of these molecules was studied as a function of sodium ion concentration and pressure. From these data we calculated the volume change accompanying the transition. Model-dependent (van't Hoff) transition parameters such as Δ H vH and transition volume (Δ V) were estimated from the analysis of conformational transitions. Experiments revealed that the Δ V for denaturation of these molecules range from − 2.35 to + 6.74 cm 3 mol −1. The expansibility (ΔΔ V/ΔT) and the pressure dependence of cation release are also presented. The difference in the volume change for this transition is related to the differences in the hydration of these molecules.

Effect of Pressure on the Stability of Short DNA Hairpins

DNA hairpins are models for more complex, naturally occurring loop-containing nucleic acid structures. They consist of two distinct structural domains: a double stranded stem and a single-stranded loop that connect the two strands of the stem. Previous studies of short DNA hairpins have revealed that the base sequence of the loop and stem significantly affect the relative thermodynamic stability of the hairpin and coil conformations. We have studied the effect of hydrostatic pressure on the heat-induced helix-coil transition of short DNA hairpins in order to gain knowledge about the role of hydration in the stability of the secondary structure adopted by these self-complementary DNA molecules. We report the effect of hydrostatic pressure on the helix-coil transition temperature (TM), monitored by absorption, for eleven 16-base DNA hairpins at different sodium chloride concentrations. The studied hairpins form by intramolecular folding of 16-base self-complementary DNA oligodeoxyribonucleotides. Each of the hairpins structures has a six-base pair duplex-forming stem linked by a four-base loop. We have varied only loop sequence and identity of the duplex base pair adjacent to the loop; the first four base pairs in the stem are the same for every molecule. Transition parameters based on a two-state denaturation process, such as ΔHvH and transition volume (ΔV), were calculated from the optical melting transitions. Experiments revealed the ΔV for denaturation of these molecules range from −2.35 to +6.74 cm3mol−1. We propose that the sequence dependence of the molar volume changes is attributable to differences in the hydration of these molecules.

Critical Exponents of Kappa Carrageenan in the Coil-Helix and Helix-Coil Hysteresis Loops

The steady-state fluorescence technique was used to study coil-helix (sol-gel) and helix-coil (gel-sol) transitions of the kappa carrageenan-water system with various carrageenan contents. Fluorescence (I) and scattered light (Isc ) intensities were measured against temperature to determine critical phase transition temperatures and exponents. It was observed that the coil-helix transition temperatures, Tch were much lower than the helix-coil (Thc ) transition temperatures due to the hysteresis of the phase transition loops. The gel fraction exponent (β) was measured and found to be in accord with the classical Flory-Stockmayer model.

DSC Study of Collagen in Disc Disease

Differential scanning calorimetry (DSC) has been used to estimate the effect of disc disease on the collagen helix-coil transition and morphology for tissue extracted from patients during surgical operation. Forty discs were obtained from patients with degenerative disc disease undergoing surgery for low back pain. The patients were in the age between 20 and 70 years old. The specimens were kept wet during DSC experiment. The data allow the comparison between thermal stability of collagen tissue from healthy patients and from patients suffering from disc disease. In the paper the comparison between thermal helix-coil transition for collagen fibers from patients suffering from disc disease and collagen fibers from healthy organisms has been discussed. The heating rate has an influence on the position on denaturation temperatures of collagen in disc tissues. Higher helix-coil transition temperature of collagen in degenerated disc suggests that additional intermolecular cross linking of collagen fibers occurs. Denaturation temperatures of collagen in degenerated male disc possess smaller values than in female ones. Disc disease induces changes in collagen structure and leads to formation of additional crosslinks between collagen fibers.

Helix−Coil Transition of DNA Monitored by Pressure Perturbation Calorimetry

We report the first use of pressure perturbation calorimetry (PPC) to characterize the heat-induced helix-coil transition of DNA polymers. The alternating copolymer poly[d(A-T)] was studied in aqueous solutions containing 5.2 and 18.2 mM Na+; it exhibited helix-coil transition temperatures of 33.6 and 44.7 degrees C, respectively. The transition is accompanied by a negative molar volume change, DeltaV) -2.6 and -2.1 mL/mol (base pair), respectively, and an increase in the coefficient of thermal expansion, Deltaalpha=+5x10(-4) K(-1) (at both ionic strengths). These values are consistent with a greater hydration of the coil form. The larger water-accessible surface area of the coil causes more water molecules to assume a bound, more densely packed structure that then gradually decreases with increasing temperature, leading to a larger value of R. The magnitude of the volume changes detected by PPC were larger than those deduced from high-pressure UV spectroscopy, shedding light on the effect of pressure on DeltaV. The shape of the PPC peak was nearly identical to the shape of the DSC peak, providing direct evidence for the correlation between the molar volume change and enthalpy change for the helix to coil transition of DNA.

Osmotically Induced Helix-Coil Transition in Poly(Glutamic Acid)

Protein folding and conformational changes are influenced by protein-water interactions and, as such, the energetics of protein function are necessarily linked to water activity. Here, we have chosen the helix-coil transition in poly(glutamic acid) as a model system to investigate the importance of hydration to protein structure by using the osmotic stress method combined with circular dichroism spectroscopy. Osmotic stress is applied using poly(ethylene glycol), molecular weight of 400, as the osmolyte. The energetics of the helix-coil transition under applied osmotic stress allows us to calculate the change in the number of preferentially included water molecules per residue accompanying the thermally induced conformational change. We find that osmotic stress raises the helix-coil transition temperature by favoring the more compact α-helical state over the more hydrated coil state. The contribution of other forces to α-helix stability also are explored by varying pH and studying a random copolymer, poly(glutamic acid- r-alanine). In this article, we clearly show the influence of osmotic pressure on the peptide folding equilibrium. Our results suggest that to study protein folding in vitro, the osmotic pressure, in addition to pH and salt concentration, should be controlled to better approximate the crowded environment inside cells.

Side-Chain and Backbone Ordering in Homopolymers

In order to study the relation between backbone and side-chain ordering in proteins, we have performed multicanonical simulations of deka-peptide chains with various side groups. Glu(10), Gln(10), Asp(10), Asn(10), and Lys(10) were selected to cover a wide variety of possible interactions between the side chains of the monomers. All homopolymers undergo helix-coil transitions. We found that peptides with long side chains that are capable of hydrogen bonding, i.e., Glu(10), and Gln(10), exhibit a second transition at lower temperatures connected with side-chain ordering. This occurs in the gas phase as well as in solvent, although the character of the side-chain structure is different in each case. However, in polymers with short side chains capable of hydrogen bonding, i.e., Asp(10) and Asn(10), side-chain ordering takes place over a wide temperature range and exhibits no phase transition-like character. Moreover, non-backbone hydrogen bonds show enhanced formation and fluctuations already at the helix-coil transition temperature, indicating competition between side-chain and backbone hydrogen bond formation. Again, these results are qualitatively independent of the environment. Side-chain ordering in Lys(10), whose side groups are long and polar, also takes place over a wide temperature range and exhibits no phase transition-like character in both environments. Reasons for the observed chain length threshold and consequences from these results for protein folding are discussed.

Temperature Dependence of the Volumetric Parameters of Drug Binding to Poly[d(A-T)]·Poly[d(A-T)] and Poly(dA)·Poly(dT)

We report the temperature and salt dependence of the volume change (Δ V b) associated with the binding of ethidium bromide and netropsin with poly(dA)·poly(dT) and poly[d(A-T)]·poly[d(A-T)]. The Δ V b of binding of ethidium with poly(dA)·poly(dT) was much more negative at temperatures ∼70°C than at 25°C, whereas the difference is much smaller in the case of binding with poly[d(A-T)]·poly[d(A-T)]. We also determined the volume change of DNA-drug interaction by comparing the volume change of melting of DNA duplex and DNA-drug complex. The DNA-drug complexes display helix-coil transition temperatures ( T m) several degrees above those of the unbound polymers, e.g., the T m of the netropsin complex with poly(dA)poly(dT) is 106°C. The results for the binding of ethidium with poly[d(A-T)]·poly[d(A-T)] were accurately described by scaled particle theory. However, this analysis did not yield results consistent with our data for ethidium binding with poly(dA)·poly(dT). We hypothesize that heat-induced changes in conformation and hydration of this polymer are responsible for this behavior. The volumetric properties of poly(dA)·poly(dT) become similar to those of poly[d(A-T)]·poly[d(A-T)] at higher temperatures.

Ribosome exit tunnel can entropically stabilize α-helices

Several experiments have suggested that newly synthesized polypeptide chains can adopt helical structures deep within the ribosome exit tunnel. We hypothesize that confinement in the roughly cylindrical tunnel can entropically stabilize alpha-helices. The hypothesis is validated by using theory and simulations of coarse-grained off-lattice models. The model helix, which is unstable in the bulk, is stabilized in a cylindrical cavity provided the diameter (D) of the cylinder exceeds a critical value D*. When D < D* both the helical content and the helix-coil transition temperature (T(f)) decrease abruptly. Surprisingly, we find that the stability of the alpha-helix depends on the number (N) of amino acid residues. Entropic stabilization, as measured by changes in T(f), increases nonlinearly as N increases. The simulation results are in quantitative agreement with a standard helix-coil theory that takes into account entropy cost of confining a polypeptide chain in a cylinder. The results of this work are in qualitative accord with most of the findings of a recent experiment in which N-dependent ribosome-induced helix stabilization of transmembrane sequences was measured by fluorescence resonance energy transfer.

A Monte Carlo simulation study of the mechanical and conformational properties of networks of helical polymers. Part II. The effect of temperature

In a recent article (Carri GA, Batman R, Varshney V, Dirama TE. Polymer 2005;46:3809 [17]) we presented a model for networks of helical polymers. In this article we generalize our results to include the effect of temperature and focus on the mechanical, conformational and thermo-elastic properties of the network. We find that the non-monotonic stress–strain behavior observed at constant temperature also appears in the stress–temperature behavior at constant strain. The origin of this behavior is traced to the induction and melting of helical beads by the application of large strains or reduction in temperature. Other conformational properties of the polymer strands are also discussed. We also study the network entropy and heat capacity, and find a non-monotonic dependence on temperature and strain. Our study shows that the entropy is controlled by the helical content whenever the latter is significant. Otherwise, the entropy corresponds to the one of a network made of random coils. In addition, the study of the heat capacity shows that strain shifts the helix-coil transition temperature significantly. Other results are also discussed.

Effect of β‐Sheet Crystals on the Thermal and Rheological Behavior of Protein‐Based Hydrogels Derived from Gelatin and Silk Fibroin

Novel protein-based hydrogels have been prepared by blending gelatin (G) with amorphous Bombyx mori silk fibroin (SF) and subsequently promoting the formation of beta-sheet crystals in SF upon exposure to methanol or methanol/water solutions. Differential scanning calorimetry of the resultant hydrogels confirms the presence and thermoreversibility of the G helix-coil transition between ambient and body temperature at high G concentrations. At low G concentrations, this transition is shifted to higher temperatures and becomes progressively less pronounced. Complementary dynamic rheological measurements reveal solid-liquid cross-over at the G helix-coil transition temperature typically between 30 and 36 degrees C in blends prior to the formation of beta-sheet crystals. Introducing the beta-sheet conformation in SF stabilizes the hydrogel network and extends the solid-like behavior of the hydrogels to elevated temperatures beyond body temperature with as little as 10 wt.-% SF. The temperature-dependent elastic modulus across the G helix-coil transition is reversible, indicating that the conformational change in G can be used in stabilized G/SF hydrogels to induce thermally triggered encapsulant release.

Comparison of the Heat- and Pressure-Induced Helix−Coil Transition of Two DNA Copolymers

The helix-coil transition of poly[d(I-C)] and poly[d(A-T)] was studied as a function of hydrostatic pressure, temperature, and sodium ion concentration. These studies were undertaken in light of a recently published phase diagram for double stranded nucleic acids [Dubins et al. J. Am. Chem. Soc. 2001, 123, 9254-9259]. The sign and magnitude of the volume change for the heat-induced helix-coil transition, DeltaV(T), of poly[d(I-C)] and poly[d(A-T)] were dependent on the helix-coil transition temperature, T(M), at atmospheric pressure. The sign of DeltaV(T) changed from negative to positive as T(M) was increased by increasing the sodium ion concentration. For poly[d(I-C)], DeltaV(T) = 0 cm(3) mol(-1), when the sodium ion concentration is such that the spectroscopically monitored T(M) = 55 degrees C at atmospheric pressure. For poly[d(A-T)], the value of DeltaV(T) = 0 under conditions such that T(M) = 47 degrees C at atmospheric pressure. Negative values of DeltaV(T) imply that the helical form is destabilized at high pressure. Under experimental conditions where the DeltaV(T) for the transition is negative, the transition could be caused by increasing the pressure under isothermal conditions. At temperatures below T(M) measured at atmospheric pressure the midpoint of the pressure-induced helix-coil transition, P(M), decreases with increasing temperature. The volume change of the pressure-induced transitions helix-coil transition, DeltaV(P), was calculated assuming a two-state model. The magnitude of DeltaV(P) (per cooperative length) was much larger than the volume change (per base pair) measured for the heat-induced transition, DeltaV(T), calculated using the Clapeyron equation. The ratio of these two volume changes was used to calculate the cooperative length for the pressure-induced transition. This parameter depends strongly on temperature, becoming greater closer to T(M) measured at atmospheric pressure. At temperatures approaching T(M) the magnitude of the cooperative length of the pressure-induced transition is approximately twice that observed for the heat-induced transition (N(T)). On the basis of the temperature dependence of the DeltaV(T) for the two polymers the coefficient of thermal expansion of the two polymers was found to be 0.17 and 0.16 cm(3) K(-1) mol(-1) for poly[d(I-C)] and poly[d(A-T)], respectively.

Alpha-helical stabilization by side chain shielding of backbone hydrogen bonds.

We study atomic models of the thermodynamics of the structural transition of peptides that form alpha-helices. The effect of sequence variation on alpha-helix formation for alanine-rich peptides, Ac-Ala21-methyl amide (A21) and Ac-A5 (AAARA)3A-methyl amide (Fs peptide), is investigated by atomic simulation studies of the thermodynamics of the helix-coil transition in explicit water. The simulations show that the guanidinium group in the Arg side chains in the Fs peptide interacts with the carbonyl group four amino acids upstream in the chain and desolvates backbone hydrogen bonds. This desolvation can be directly correlated with a higher probability of hydrogen bond formation. We find that Fs has higher helical content than A21 at all temperatures. A small modification in the amber force field reproduces the experimental helical content and helix-coil transition temperatures for the Fs peptide.

Effects of carrageenan type on the behaviour of carrageenan/milk mixtures

The apparent hydrodynamic diameter of casein micelles was determined as a function of hydrocolloid concentration at 60°C and during cooling to 20°C. The systems studied contained skim milk 100-fold diluted in permeate in the presence of either lambda-, iota-, kappa-carrageenan or guar gum. These measurements show that all three forms of carrageenan adsorb onto casein micelles, whereas guar gum does not. Lambda-carrageenan (which is always in the coil conformation) adsorbs at all the temperatures studied, whereas the iota and kappa forms adsorb only at temperatures below the onset of the helix–coil transition. The results agree with the idea that adsorption of carrageenan occurs only above a certain minimum charge density. Phase diagrams were established at 60°C for the three carrageenans and the rheological behaviour was followed on cooling and at 25°C. The results suggest that the lambda-carrageenan bridges the casein micelles at low carrageenan concentrations, leading to the sedimentation of carrageenan/milk mixtures at 60°C and structure formation on cooling. At 60°C, iota- and kappa-carrageenan induce depletion flocculation of casein micelles above a critical carrageenan concentration. On cooling, systems containing iota-carrageenan clearly form a network at the helix–coil transition temperature. The network is formed by mixed carrageenan/casein micelle crosslinks. In the presence of excess carrageenan, this network is reinforced by carrageenan/carrageenan crosslinks. For the kappa-carrageenan/casein micelle mixtures the existence of mixed carrageenan/casein micelle crosslinks is less obvious but seems likely.

α-Helix propensities of homo-oligomers in aqueous solution studied by multicanonical algorithm

Helix–coil transitions of homo-oligomers in aqueous solution are studied by multicanonical Monte Carlo simulations. The solvation effects are represented by the term that is proportional to the solvent-accessible surface area of the peptides. Homo-oligomers of length 10 are considered for two characteristic amino acids, alanine and glycine, which are helix former and helix breaker, respectively. It is shown that the helix–coil transition temperature for homo-alanine in water is considerably lower than in gas phase. The helix propagation parameter s and nucleation parameter σ for both alanine and glycine are calculated and shown to be in remarkable agreement with experimental results.

Chain length and oligonucleotide stability at high pressure.

The effect of hydrostatic pressure on the helix-coil transition temperature (Tm) was measured for the DNA oligomers (dA)n(dT)n, where n = 11, 15, and 19, in 50 mM NaCl. The data were analyzed in light of previously published data for the polymer, poly(dA) center dot poly(dT) under the same conditions. As has been observed for DNA polymers, increasing the hydrostatic pressure led to an increase in the Tm of the oligomers; however, the effect of pressure diminished with decreasing chain length. The value of dTm/dP decreased linearly with the inverse of the chain length varying from 3.15 x 10(-2) degrees C MPa-1 for the polymer to 0.7 x 10(-2) degrees C MPa-1 for the 11-mer. The two-state or van't Hoff enthalpy (DeltaHvH) of the helix-coil transition was obtained by analysis of the half-width of the thermal transition. As expected, DeltaHvH decreases with decreasing chain length. In contrast to the behavior of the polymer, poly(dA) center dot poly(dT), and (dA)19(dT)19, the DeltaHvH of the two shorter duplex oligonucleotides displayed a small pressure dependence dDeltaHvH/dP approximately equal -0.4 kJ MPa-1 in both cases. The changes observed in the Tm and DeltaHvH were not sufficient to explain the magnitude of the chain-length dependence of the pressure effect. To interpret the large chain-length dependence of dTm/dP, we propose that the terminal base pairs contribute a negative volume change to the helix-coil transition. Base pairs distant from the ends exhibit behavior characterized by the polymer where end effects are assumed to be negligible, i.e., a positive volume change for the helix-coil transition. The negative volume change of separating terminal bases may originate from the imperfect interactions these base pairs form with water due to the existence of several energetically equivalent conformations. This is reminiscent of one of the mechanisms proposed to be important in the pressure-induced dissociation of multimeric proteins into their constituent subunits.